This project is focused on developing a novel tool for studying gut microbiome impact on human health: an in vitro human model of gut epithelium-microbiome-immune homeostasis. While the gut microbiome is known to have tremendous impact on human health, these effects are complex and generally not well understood, limiting translation of observed microbiome impact to effective therapies. There is currently no in vitro model of human gut epithelium-microbiome-immune homeostasis, and most studies of these effects are thus currently carried out in gnotobiotic rodent models. A controlled, defined in vitro human system capturing key gut microbiome-epithelium-immune interactions would thus be a tremendously valuable tool to academic and industrial scientific and medical research communities. The approach combines development of the necessary hardware as well as culture protocols and biomaterials required for primary intestinal-immune mammalian culture and establishment of microbial populations to be cultured with gut mammalian cells and representative of gut commensal microbe populations. Importantly, the project team has already worked together to establish base fluidic culture platforms and immune-competent biological gut models with co-cultured simple microbial communities, and thus has demonstrated the ability to utilize their unique combination of complementary expertise and move projects toward commercialization. Specific hardware to be developed (Aim 1) includes a fluidic platform for flow mimicking both the circulatory system and flow of gut luminal contents, highly important for controlling oxygen concentration and maintenance of gut homeostasis with a resident microbial population, as well as sensors for oxygen and intestinal barrier function (trans-epithelial electrical resistance, TEER). The primary intestinal culture system (Aim 2) will be in the form of a monolayer for facile access to the apical mucosal surface and will include dendritic cells for immune function. PEG-based biomaterials with tunable chemical functionality and mechanical properties will be used to support the primary intestinal culture, including incorporation of cells key to maintenance of intestinal homeostasis: myofibroblasts and enteric glia. Microbial consortia (Aim 3) will be developed from isolated strains from human samples, and screened for maintenance of homeostasis in gut culture. The integrated gut epithelium-microbiome-immune biological system will be maintained on the developed fluidic platform for extended (2 week) culture, and the impact of the commensal populations on metrics of gut viability and function, as well as cytokine release profiles providing insight into cell signaling events will be analyzed. Tw case studies will be used to assess the ability of the resulting hardware-biology composite system to capture well-characterized responses of the human microbiome (Aim 4). The hardware-culture system will be stimulated to induce leaky gut and inflammation, which will then be ameliorated with specific bacterial species demonstrated to provide beneficial effects in these conditions.